Method for identifying a mammal receptive to embryo transfer

ABSTRACT

The present invention is drawn to novel methods of selecting mammals receptive to embryo transfers in order to improve reproductive efficiency and milk production of the mammals. The present invention encompasses three methods: (1) selecting from any group of mammals; (2) selecting from estrus-negative mammals; and (3) selecting from estrus-negative mammals and pooling the selected mammals with estrus-positive mammals. Also encompassed is a kit for selecting mammals receptive to embryo transfers.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Division of U.S. application Ser. No. 13/495,656,filed Jun. 13, 2012, which itself issued as U.S. Pat. No. 10,478,460 onNov. 19, 2019, which claims the benefit of priority to U.S. provisionalapplication No. 61/496,761, filed Jun. 14, 2011, the entire contents ofeach of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present technology relates to the identification of reproductivemammals that, prior to the inventive technology, would have beenclassified as unreceptive to embryo transfer and artificial inseminationmethods for inducing pregnancy.

BACKGROUND

In recent years, the dairy industry has seen a drastic decrease in thereproductive efficiency of cows. This is reflected in undesirabledecreases in such industry-standard reproductive performancemeasurements as (1) days to first service, (2) days to conception, (3)calving interval, (4) services per conception, (5) non-return rate, (6)estrus (or “heat”) detection rate, and (7) pregnancy rate. Estrus, orheat, detection, in particular, is useful for identifying cows that arelikely to become pregnant if inseminated at the time of estrusdetection, or shortly thereafter. Wrongly identifying a cow as not being“in heat” (i.e., “estrus-negative”) is a significant contributor toreproductive inefficiency. This has a corresponding significant impacton the dairy industry because dairy cows that are not reproductivelyefficient are culled.

SUMMARY

One aspect of the present invention is a method of selecting mammalsreceptive to embryo transfer, comprising: (1) synchronizing the estrouscycles of at least two mammals; (2) from the at least two mammals instep (1), selecting a mammal that has a corpus luteum (CL) present inits ovary; (3) transferring an embryo to the mammal selected in step(2); and (4) testing receptivity of the mammal towards pregnancy.

Thus, in one embodiment, the present invention provides a method foridentifying a mammal receptive to embryo transfer, comprising (A)synchronizing the estrous cycles of two or more mammals; (B)transferring an embryo to a mammal that has a corpus luteum but has beenidentified as not in estrus or estrus-negative; and (C) testing themammal for pregnancy, wherein a pregnant mammal indicates the mammal'sreceptivity to embryo transfer.

In one embodiment, the mammals are cows. In another embodiment,synchronizing is achieved by administering prostaglandin to the at leasttwo mammals. In another embodiment, synchronizing is achieved byadministering one or more of prostaglandin, gonadotropin and progestin.

In another embodiment, the mammals are cows and the presence of a corpusluteum is detected by a method selected from the group consisting of (i)rectal palpation; (ii) ultrasonography; (iii) measuring theconcentration of progesterone in the blood of the cow; and (iv)measuring the concentration of the progesterone in the milk produced bythe cow.

In another embodiment, the mammals are cows and the embryo is between 5days and 10 days old.

In another embodiment, the mammals are “estrus-negative”, i.e., themammals are not “in heat or do not show outward signs of heat.” In afurther embodiment, the at least two estrus-negative mammals are cows.

In another embodiment, the synchronizing is achieved by administeringprostaglandin to the at least two estrus-negative cows. Theprostaglandin may be used in conjunction with other pharmaceuticals in asynchronization program.

In another embodiment, wherein the presence of a corpus luteum in the atleast two estrus-negative cows is detected by a method selected from thegroup consisting of (i) rectal palpation; (ii) ultrasonography; (iii)measuring the concentration of progesterone in the blood of the cow; and(iv) measuring the concentration of the progesterone in the milkproduced by the cow.

In a further embodiment, the embryo to be transferred to the at leasttwo estrus-negative cows is between 5 days and 8 days old.

In a further embodiment, the embryos to be transferred are freshembryos, frozen embryos, vitrified embryos, in vivo embryos, in vitrofertilized embryos, embryos produced by somatic cell nuclear transfer,embryos produced by embryonic cell nuclear transfer, embryos produced byembryonic splitting, embryos produced by sperm microinjection, embryosproduced by genetic modification, and/or embryos produced by geneticselection.

In a further embodiment, the mammal to receive the embryo is in theestrous cycle between day 6 and day 10.

An additional embodiment comprises detecting whether the mammals are inestrus or “in heat”, so as to identify estrus-positive andestrus-negative mammals.

An additional embodiment comprises detecting whether an ovarian corpusluteum is present in the mammals that have been identified as not inestrus or estrus-negative.

Another aspect of the invention is a method for selecting mammalsreceptive to embryo transfers, comprising: (1) synchronizing the estrouscycles of at least two mammals; (2) from the at least two mammals instep (1), selecting a mammal that is estrus-negative; (3) from themammal in step (2), selecting a mammal that has a corpus luteum (CL)present in its ovary; (4) transferring an embryo to the mammal selectedin step (3); and (5) testing receptivity of the mammal towardspregnancy.

In one embodiment, the method for selecting mammals receptive to embryotransfers further comprises between step (1) and (2) selecting anestrus-positive mammal from the at least two mammals, wherein an embryois transferred to the estrus-positive mammal and the CL-bearing mammalselected in step (3) In another embodiment, the at least two mammals arecows.

In one embodiment, the method for selecting mammals receptive to embryotransfers further comprises between step (1) and (2) selecting anestrus-positive mammal from the at least two mammals, wherein an embryois transferred to the estrus-positive mammal and the CL-bearing mammalselected in step (3) and wherein the milk production from theestrus-positive mammal and the CL-bearing mammal is at least 1% higherthan the milk production from the estrus-positive mammal alone. In thisregard, the milk production from the estrus-positive mammal and theCL-bearing mammal can be 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%,30-35%, 35-40%, 40-45%, 45-50% higher than the milk production from theestrus-positive mammal alone.

In another embodiment, the estrus-positive cows are selected by a methodselected from the group consisting of: (i) tail marking; (ii) watchingestrus signs; (iii) using marker animals; (iv) mounting-activitydetectors; (v) measuring methyl heptanol and (vi) measuring pheromone(vii) measuring motion (viii) measuring temperature.

In another embodiment, the at least two mammals are cows and thesynchronizing is achieved by administering prostaglandin to the at leasttwo mammals. In another embodiment, the synchronizing is achieved byadministering one or more of prostaglandin, gonadotropin and progestinto the cows.

In another embodiment, the at least two mammals are cows and thepresence of a corpus luteum is detected by a method selected from thegroup consisting of: (i) rectal palpation; (ii) ultrasonography; (iii)measuring the concentration of progesterone in the blood of the cow; and(iv) measuring the concentration of the progesterone in the milkproduced by the cow.

In yet another embodiment, the embryo to be transferred to the at leasttwo cows is between 5 days and 10 days old. In yet another embodiment,the embryos to be transferred are fresh embryos, frozen embryos,vitrified embryos, in vivo embryos, in vitro fertilized embryos, embryosproduced by somatic cell nuclear transfer, embryos produced by embryoniccell nuclear transfer, embryos produced by embryonic splitting, embryosproduced by sperm microinjection, embryos produced by geneticmodification, and/or embryos produced by genetic selection.

In yet a further embodiment, the cow to receive the embryo is in theestrous cycle between day 6 and day 10.

Another aspect of the invention is a kit to increase reproductiveefficiency of mammals, comprising: (1) at least one estrus-detectingreagent or device, (2) at least one corpus luteum (CL)-detecting reagentor device, (3) at least one warming or thawing medium and (4) at leastone transfer medium.

In one embodiment, the at least one estrus-detecting device is amounting-activity detector or motion activity detector. In anotherembodiment, the at least one CL-detecting reagent comprises an antibodyagainst progesterone and an antibody against the antibody againstprogesterone.

Another aspect of the invention is a kit to impregnate one or moreestrus-negative cows, comprising: (1) at least one corpus luteumdetecting reagent or device; and (2) at least one embryo to betransferred to at least one cow that has been identified to have acorpus luteum by said corpus luteum detecting reagent or device.

In one embodiment, wherein the corpus luteum detecting reagent comprisesan antibody against progesterone and an antibody against the antibodyagainst progesterone.

In another embodiment, the embryos to be transferred are fresh embryos,frozen embryos, vitrified embryos, in vivo embryos, in vitro fertilizedembryos, embryos produced by somatic cell nuclear transfer, embryosproduced by embryonic cell nuclear transfer, embryos produced byembryonic splitting, embryos produced by sperm microinjection, embryosproduced by genetic modification, and/or embryos produced by geneticselection.

Another aspect of the invention is a method of increasing milkproduction, comprising: (1) synchronizing the estrous cycles of at leasttwo mammals; (2) from the at least two mammals in step (1), selecting amammal that is estrus-negative; (3) from the mammal in step (2),selecting a mammal that has a corpus luteum (CL) present in its ovary;(4) transferring an embryo to the mammal selected in step (3); and (5)testing receptivity of the mammal towards pregnancy.

In one embodiment, the method of increasing milk production furthercomprising synchronizing the estrus cycles of the cows.

In another embodiment, the synchronization is achieved by administeringprostaglandin to the cows. In another embodiment, the synchronizing isachieved by administering one or more of prostaglandin, gonadotropin andprogestin to the cows.

In another embodiment of the method of increasing milk production, thepresence of a corpus luteum is detected by a method selected from thegroup consisting of: (i) rectal palpation; (ii) ultrasonography; (iii)measuring the concentration of progesterone in the blood of the cow; and(iv) measuring the concentration of the progesterone in the milkproduced by the cow.

In yet another embodiment of the method of increasing milk production,the embryo is between 5 days and 10 days old. In yet another embodiment,the embryos to be transferred are fresh embryos, frozen embryos,vitrified embryos, in vivo embryos, in vitro fertilized embryos, embryosproduced by somatic cell nuclear transfer, embryos produced by embryoniccell nuclear transfer, embryos produced by embryonic splitting, embryosproduced by sperm microinjection, embryos produced by geneticmodification, and/or embryos produced by genetic selection.

In yet a further embodiment, the cow to receive the embryo is in theestrous cycle between day 6 and day 10.

A further aspect of the invention is a method for improving thepregnancy rate of a cow population, comprising: transferring an embryointo a cow that has been identified as estrus-negative, and wherein theestrus-negative cow has been synchronized by administering one or moreof prostaglandin, gonadotropin and progestin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Conception rates for animals in each synchrony group (notdifferent to one another). (P>0.1).

FIG. 2: Conception rates for animals in known and unknown synchronygroups were found not to be different. (P>0.1)

FIG. 3: Pregnancy rates for beef cows, including estrus-positive cows asidentified by “standing heat”, estrus-positive cows as identified by“tail chalk”, and estrus-negative but CL-positive cows as identified byCL palpation.

FIG. 4: Pregnancy rates for repeated breeder cows from Dairy A,including estrus-positive cows as identified by “tail chalk”, andestrus-negative but CL-positive cows as identified by CL palpation.

FIG. 5: Pregnancy rates for estrus-positive cows as identified by“standing heat”, estrus-positive cows as identified by “tail chalk”,estrus-negative but CL-positive cows as identified by CL palpation, andcows subjected to timed embryo transfer.

DETAILED DESCRIPTION

Maximizing the number of cows in a herd that can become pregnant—andtherefore begin lactating to produce milk—is a chief goal among dairyfarmers. In the dairy industry, monitoring the pregnancy rate of cows isa standard way to measure the reproductive performance of individualcows. And the parameter of pregnancy rate is calculated as theconception rate multiplied by estrus (“in heat”) detection rate. In theestrus phase of the estrous cycle, females become sexually receptive andare often observed as “in heat.” Each cow may experience seventeenestrous cycles per year if they do not become pregnant and they residein locations where adequate light and nutrition is available year round.The estrous cycle generally consists of four phases: (1) proestrus; (2)estrus; (3) metestrus and (4) diestrus. The corpus luteum is a structurethat often protrudes from the edge of the ovary and can bedifferentiated from the follicle by palpation or ultrasound. The ovarianfollicle usually contains a single oocyte and is found at the cortex, orouter part, of the ovary. The corpus luteum, which derives from ovarianfollicles, is also located at the ovarian cortex. Developmentally,during each estrus phase, ovarian follicles develop into maturity underthe influence of estrogen and gonadotropic hormones. For some mammalssuch as cows, spontaneous ovulation may occur in the estrus phase, whilein other species, ovulation requires sexual intercourse.

With respect to cows, and the calculation of pregnancy rates, the heatdetection rate is determined by the fraction of cows that are identifiedas being in estrus during a 21 day period, via various methods discussedin methods below. An important point is that variability in correct heatdetection can have a dramatic effect on the pregnancy rate of thecollective herd. For instance, if the conception rate is 40%, and theheat detection rate is identified as 50%, then the pregnancy rate wouldequal 20% (PR=0.4*0.5=0.2). However, if all cows were correctlyidentified as being in heat, or in estrus, then the pregnancy rate woulddouble to 40% (PR=0.4*1.0=0.4). Consequently, misidentifying the numbersof cows that could actually become pregnant by artificial inseminationor embryo transfer, but which were otherwise culled because “no heat”was detected, can significantly reduce the productivity of the entireherd. In addition, incorrect identification of estrus, and breeding cowsthat are not in estrus, costs the dairymen for the wasted semen and losttime while waiting to determine if the animal is pregnant. Therefore,breeding all animals synchronized is also costly.

The present invention maximizes the number of cows selected from a herdto be recipients for embryo transfer by identifying additional animalsnot seen in estrus “estrus-negative’ that are still receptive to embryotransfer, and thus pregnancy. “Receptive” typically refers to thehormone-induced, thickened condition of the uterine lining that providesnourishment for the implanted embryo. The present invention allows dairyproducers to impregnate cows that had not shown signs of estrus.Although these cows were capable of becoming pregnant they would nothave been identified using other protocols. In addition, this newprotocol also provides a superior method for impregnating cows over thestandard timed artificial insemination method, because it can permitfewer shots and fewer drugs to be administered to cows over time.

Artificial insemination refers to the introduction of semen into thevagina or uterus by mechanical or instrumental means rather than bysexual intercourse. Artificial insemination is planned to coincide withthe expected time of ovulation so that fertilization can occur. Timedartificial insemination protocols are designed to synchronize ovulationamong a group of cows that are often in various stages of the estrouscycle at the initiation of the program. In short, the present inventionprovides an alternative and preferred method to increase pregnancies andtherefore lifetime milk production.

The present invention identified that cows that were classified asestrus-negative, i.e., cows that were not identified as in heat, butwhich had a corpus luteum in the ovary, were in fact receptive to embryotransfer, and could become pregnant. Thus, the present inventive methodpermits the identification of reproductively sound cows from thesubpopulation of cows in the herd that would otherwise have been culled.That is, normally, a dairy farmer will segregate and cull or first useother strategies such as timed AI to increase the number of pregnanciesand then cull. Typically they would only use those identified as beingin heat for embryo transfer or artificial insemination to becomepregnant. Many reproductively sound cows, particularly on dairy's, donot show heat because of things like poor footing, negative energybalance, excessive heat and inadequate ventilation, inadequate space,inadequate bedding etc. and therefore have a silent or estrus-negativeheat. By using the present inventive method, however, the dairy farmercan increase the reproductive efficiency of the cattle herd byrecategorizing the to-be-culled subpopulation so as to identifyestrus-negative cows that nonetheless are still able to become pregnant.Thus, any variability in heat detection can be corrected for bydetecting the presence of corpus luteum in the estrus-negative cows,allowing more cows per herd to be used for reproduction, and thus milkproduction.

Accordingly, one aspect of the present inventive method is identifyingthe presence of a corpus luteum in estrus-negative cows. The presentinvention does not require an initial detection of estrus, ornon-detection of estrus, as the case may be. Rather, the presentinvention also accommodates simply the detection of corpus luteum in allcows regardless of whether or not they have been subjected toestrus/heat detection. Furthermore, the present invention is not limitedto use only in cows. Other female mammals can be subjected to theinventive test to determine whether they are suitably biologicallyreceptive for reproductive activities. Thus, other animals, includinghorses, dogs, cats, sheep, goats, camelids, pachyderms, as well asothers, can be tested for the presence of a corpus luteum, or identifiedas being in heat, or not in heat, and the latter subsequently tested forthe presence of a corpus luteum prior to application of reproductivetechnology, such as by embryo transfer. Thus, citation to “cows” as usedelsewhere herein is only an illustrative example, and not meant to beinterpreted as limiting the inventive method to only that species ofanimal.

Thus, in one embodiment, a subpopulation of estrus-negative cows isprovided that are nonetheless receptive to embryo transfer. Thisparticular embodiment therefore concerns identifying those cows from aherd that are not seen in heat, and then testing those estrus-negativecows to determine which contain a corpus luteum (CL). Theseestrus-negative, CL-positive cows are then implanted with embryos, and,of that subpopulation, some will become pregnant. Accordingly, presentinvention combined with appropriate synchronization increases thereproductive efficiency of the herd.

In this regard, the corpus luteum is a temporary endocrine structuredeveloped from an ovarian follicle during the metestrus and diestrusphases of the estrous cycle in mammals. The corpus luteum produces highlevels of progesterone, which thicken and maintains the uterine liningin preparation for implantation of an embryo. In the absence of a viableembryo, the corpus luteum stops secreting progesterone and regressesinto a scar tissue, while the uterine lining consequently degeneratesback to normal size. With the presence of a viable embryo, however, thecorpus luteum continues to secrete progesterone, which in turn maintainsthe thick uterine lining to provide the necessary nourishment for thedevelopment of the embryo.

There are three standard ways to detect the corpus luteum: (1) rectalpalpation; (2) ultrasonography; and (3) measuring progesterone levels inthe blood or milk produced by the mammal. In the case of rectalpalpation in cows, the cervix is first typically detected by hand, touchand feel in order to identify the uterus, which is characterized by anexternal bifurcation between the two uterine horns. The next step is todetect the ovaries, which are typically located underneath or to theside of the uterus. The corpus luteum is a structure that oftenprotrudes from the edge of the ovary and can be differentiated from thefollicle by palpation or ultrasound. The ovarian follicle contains asingle oocyte and is found at the cortex, or outer part, of the ovary.The corpus luteum, which derives from ovarian follicles, is also locatedat the ovarian cortex.

When performing ultrasonography, a probe is inserted through the rectumand positioned above the ovary. Pulses of ultrasound are generated bythe probes and transmitted to the tissues adjacent to the probe. Fromthe adjacent tissues, the pulses of ultrasound are reflected back to theprobe. The amount of the reflection depends on the structure of theadjacent tissues. The corpus luteum is typically seen as a distinctgrayish echogenic area within the ovarian stroma and could contain acavity. Ultrasonography offers a highly reliable way of detecting corpusluteum, however, equipment is costly.

Lastly, the corpus luteum can be detected by measuring the concentrationof the hormone progesterone in the cow's blood or milk. Assay kits arecommercially available to determine progesterone level in blood or milk.For example, a “Target Equine Progesterone Kit” is available fromBioMetallics Incorporated, Princeton, N.J., USA. A QUICKCHECK™ CanineProgesterone Kit and a QUICKCHECK™ Equine Progesterone Kit are availablefrom Exodus Breeders Corporation in York, Pa., USA.

As mentioned above, the present inventive method permits theidentification of cows containing a corpus luteum as suitable candidatesfor embryo transfer regardless of whether or not the cows have beenclassified as in heat or not in heat.

There are various traditional methods for determining whether a cow isin heat. Mammals in heat, i.e., that are in the estrus phase of theestrous cycle, usually display certain estrus signs. In the case ofcows, these signs include: (1) standing to be mounted by another cow,which is the most commonly used sign in the industry for estrusdetection; (2) greater physical activity as a result of increasednervousness and restlessness; (3) licking and sniffing of the genitalareas of other cows; (4) bawl or bellow frequently; (5) a thin, clear,watery mucus discharge from vulva; and (6) reduced feed intake. Becausemany of these individual signs require human observation and even thencan be subtle in nature, a determination of whether a cow or othermammal is in heat should be based on the totality of all signs. A mammalis deemed “estrus-positive” if it is determined that the totality of allsigns points to estrus. Conversely, a mammal is deemed “estrus-negative”if it is determined that the totality of all signs does not point toestrus.

Specifically, whether a cow is in heat or not can be identified by“standing heat” and “tail chalk”. Those in “standing heat” showedbehavioral estrus by standing to be jumped by another cow. Further,“tail chalk” can be used to identify cows in estrus—by painting the tailhead of all cows synchronized and observing whether the paint is rubbedoff. If the paint is rubbed off, the cow is recorded as having shownsign of estrus.

The dairy farmer is aware of many of the practical ways in which theaforementioned signs can be detected. “Marker animals,” for example, aresurgically-altered bulls or androgenized cows or steers, that aresexually active and seek out cows in estrus. Androgenized cows are cowsinjected with testosterone and simulate male behavior. These markeranimals are typically equipped with a marking device, such as a halterthat works like a ball-point pen under the marker mammal's chin. Whenthe marker bull mounts a cow that is in heat, the chin-ball markerleaves mark on the female mammal's back. These marks can then beinterpreted to determine whether the female mammal is in heat.Similarly, mounting-activity detectors attached to a cow can changecolor upon sexual activity, while electronic detectors transmit radiosignals to a computer that records the identity of the cow, and the timeand duration of the mount. Electronic detectors are more accurate thanother types of detectors because they can record multiple mounts and theduration of the mount. Alternatively, increased levels of methylheptanol in a cow's vaginal secretions indicates it is in heat. Methylheptanol can be monitored through gas chromatography as described, forinstance, in U.S. Pat. No. 4,467,814, which is hereby incorporated byreference in its entirety. “Tail marking” is yet another method todetect estrus by placing chalk or paint in a band along a mammal'stailhead from hooks to pins. The chalk or paint is rubbed off by themammal doing the mounting.

Accordingly, one of ordinary skill in the art would know how to detectcows that are in heat, or are not in heat, and subsequently identify inthe latter (estrus-negative) subgroup, those cows which have a corpusluteum. The cows that are estrus-negative but CL-positive then canbecome candidate recipients of embryos in embryo transfer procedures.

In this regard, embryo transfer refers to various standard methods forestablishing a pregnancy in which one or several embryos are isolatedfrom at least one donor mammal and placed into the uterus of at leastone other mammal. An in vivo embryo is one isolated from the donormammal. In addition to producing the embryo in a donor mammal, an embryocan also be produced with the process of in vitro fertilization (IVF).IVF refers to a process by which an egg is fertilized by a sperm outsidethe body. Embryos may be transferred fresh or after preservation byfreezing or vitrifying in liquid nitrogen. Frozen/vitrified embryos arethawed/warmed prior to embryo transfer following protocols dependent onthe preservation process and the tool used to contain the embryo. Frozenembryos can maintain their viability for long periods of time, likelybecause the low temperature effectively stops all biological activities,including biological reactions leading to apoptosis.

In one embodiment, the embryos to be transferred are produced in vitroby somatic cell nuclear transfer (i.e., cloning), which is exemplifiedin Kato et al., Science 282(5396):2095-2098 (1998) and Wilmut et al.,Nature 385:810-813 (1997), both of which are hereby incorporated byreference in their entireties.

In another embodiment, the embryos to be transferred are produced invitro by embryonic cell nuclear transfer, which is exemplified in Peuraet al., Mol. Reprod. Dev. 50:185-191 (1998) and Smith et al., Biology ofReproduction 40(5): 1027-1035 (1989), both of which are herebyincorporated by reference in their entireties.

In a further embodiment, the embryos to be transferred are produced invitro by embryo splitting or cleavage, which is exemplified in Gary etal., Therigenology 35(1):37-44 (1991) and Baker et al., Therigenology23(1):3-12 (1985), both of which are hereby incorporated by reference intheir entireties.

In an additional embodiment, the embryos to be transferred are producedin vitro by sperm microinjection, which is exemplified in Heuwieser etal., Therigenology 38(1):1-9 (1992), which is hereby incorporated byreference in its entirety. In one embodiment, the embryos to betransferred are produced in vitro by genetic modification, which isexemplified in Yang et al., PLoS One 6(3):e17593 (2011); Clark, Am. J.Clin. Nutr. 63:633-638 (1996); and Lai et al., Reprod. Biol. Endocrinol.1:82 (2003), all of which are hereby incorporated by reference in theirentireties.

In one embodiment, the embryos to be transferred are produced in vitroby genetic selection, which is exemplified in Bodo et al., Acta Vet.Hung. 49(1):99-109 (2001) and Chen et al., Biology of Reproduction.67(5): 1488-1492 (2002), both of which are hereby incorporated byreference in their entireties.

The embryo transfer procedure with respect to fresh or in vivodonor-generated embryos, generally comprises seven steps: (1) selectionof donor mammal; (2) synchronization of donor mammal and recipientmammals; (3) superovulation of donor mammal (a process of manipulatingthe donor mammal to produce maximum number of eggs); (4) insemination ofthe donor (fertilization of donor eggs by sperm from selected males);(5) flushing the embryo (separating the embryos from the donor uterus);65) evaluation of the embryo (classifying the embryos into differentgrades, such as excellent, good, fair, poor and dead); (7) selection ofthe recipient mammal (selecting female mammals that are receptive toembryo transfer); (8) transferring the embryos, which is a process of“loading” the embryo into a transfer gun, inserting the transfer gunthrough the cervix and then expelling the embryo in the forward tip of auterine horn ipsilateral to the corpus luteum. In the case of in vitroproduced embryo, only steps (5), (6) and (7) typically would apply.Sometimes it is desirable to select a donor mammal that is regarded tobe “genetically superior” than other members of its species. Thus, inone embodiment of the present invention, embryo transfer can be used toincrease the number of offsprings from genetically superior mammals. Forinstance, artificial insemination is a low cost alternative comparedwith maintaining bulls. Artificial insemination also provides theability to select from a large number of bulls those that haveparticularly desirable traits. Embryo transfer has been used to maximizethe reproductive potential of such elite cows that can be bred withthose bulls. With the help of hormone stimulation, for example, elitecows can be subject to superovulation to produce 7-12 embryos perovulation cycle to be transferred to other surrogate cows. At about 50%successful transfer rate, the number of calves from an elite cowtherefore increases from one to more than six per ovulation cycle. Cowscan be subject to superovulation multiple times in a year.

Various media used in embryo transfer are known in the art. For example,embryo flush can be used when removing in vivo embryos from a donor'suterus. Commercially available embryo flush include, for example,EmCare™ Complete Flush Media and Vigro™ Complete Flush Media fromReproduction Resources, as well as BoviPro™ Recovery Media from Minitubeof America. Further, holding media can be used when manipulating embryoson a benchtop. Commercially available holding media include, forexample, EmCare™ Holding Media, Vigro™ Holding Plus Media and SyngroHolding Media from Reproduction Resources, as well as BoviPro™ HoldingMedia from Minitube of America. Additionally, thawing media can be usedfor thawing frozen embryos. Commercially available thawing mediainclude, for example, EmCare™ CSU Thawing Kit from ReproductionResources, as well as BoviPro™ Rehydration Media from Minitube ofAmerica.

Before estrus detection and/or corpus luteum detection are performed, itcan sometimes be desirable to “synchronize” a herd of cows to ensuresubstantially all members of the dairy herd are staged at the same orsimilar reproductive period of their respective estrous cycles.Synchronizing the estrous cycles generally means manipulating thereproductive process of a group of female mammals so that ovulationoccurs at approximately the same time. Synchronized estrous cycles incows, for instance, allow a breeder to schedule breeding activities fora group of cows during a predetermined period, thereby increasing theefficiency of artificial insemination, embryo transfer or other breedingtechniques, useful for maximizing the reproductive efficiency of theentire herd.

Synchronization can be achieved through the application three primarygroups of hormones: prostaglandins, progestins, and gonadotropins. Thereare commercial products available for each group of hormones.Prostaglandin products include Lutalyse, Estrumate, and IN-SYNCH.Progestin products include the vaginal implant CIDR® and the feedadditive Melengestrol Acetate (MGA). Gonadotropins products includeCystrorelin, Factrel, and Fertagyl. Antibodies can be used to detectthese hormones. As is well known, an antibody is a protein employed bythe immune system to recognize and neutralize foreign objects such asbacteria and virus. The target of an antibody is termed an antigen. Theantibody typically recognize a unique part (“epitope”) of an antigen. Inaddition to bacteria and viruses, modern molecular biology techniquesnow allow a skilled artisan to raise antibodies against almost anyprotein and then use the antibody as an assay tool as well as diagnosticor therapeutic tool.

“Prostaglandin” is a group of 20-carbon fatty acid hormones, secreted bythe uterine endometrium, which causes the regression of corpus luteumand thereby induces a sharp drop of progesterone produced by the corpusluteum. With the removal of progesterone, gonadotropin-releasing hormone(GnRH) starts to exert greater influence. The higher level of GnRH leadsto higher level of follicle-stimulating hormone (FSH) and luteinizinghormone (LH), which supports follicular development as the mammal entersthe proestrus phase of the estrous cycle. “Progesterone” is a 21-carbonhormone that works through the intracellular progesterone receptor toexert a number of physiological effects, including (1) converting theendometrium, or uterine lining, to its secretory stage to prepare forthe implantation of fertilized egg; (2) decreasing maternal immunityduring implantation for the female mammal's body to accept pregnancywithout an immune reaction; (3) decreasing the contractibility of theuterine smooth muscle; and (4) inhibiting lactation during pregnancy.During the metestrus phase of the estrous cycle, the newly formed corpusluteum starts to secret low level of progesterone. The corpus luteumthen secretes high levels of progesterone during the diestrus phase.

A number of protocols can be employed to achieve synchronization, suchas administering one hormone or a combination of hormones. For instance,prostaglandin may be injected individually in one of the four ways:

(1) A single injection of prostaglandin is administered and estrus isdetected in the following 2-5 days.

(2) Estrus is detected in the first five days, and all female mammalsfound to be in heat were bred. Then a single injection of prostaglandinis administered on the fifth day to the female mammals that were not inheat.

(3) A first injection of prostaglandin is administered and estrus isdetected in the next 5 days. On the 11^(th) day, a second injection ofprostaglandin is administered to the female mammals that were not inheat.

(4) A first injection of prostaglandin is administered and estrus isdetected in the next 5 days. On the 14^(th) day, a second injection ofprostaglandin is administered to the female mammals that were not inheat.

The administration of multiple shots of prostaglandins is typicallyreferred to by a person skilled in the art as the two-shot method. SeeDuPonte M W and Lee K K, Three Simplified Estrus SynchronizationPrograms for Hawaii's Beef Breeding Season, Livestock Management(Cooperative Extension Service, University of Hawaii at Manoa), April2007, LM-14, 1-2, which is hereby incorporated by reference in itsentirety.

Prostaglandin can also be administered together with a progesterone-likefeed additive Melengestrol Acetate (MGA). MGA can be fed for 14 days andthen withdrawn. A single injection of prostaglandin then is administered17 to 19 days after the MGA withdrawal.

In one embodiment, after synchronizing the cows with a single injectionof prostaglandin, no other synchronizing agent is administered to thecows before the detection of corpus luteum. In another embodiment, aftersynchronizing the cows with a single injection of prostaglandin, noother synchronizing agent is administered to the cows before embryotransfer.

Synchronization can also be achieved through the combination ofprostaglandin and vaginal implant CIDR®. Typically the CIDR implant isinserted on Day 0, one injection of prostaglandin is given on Day 6, andthe CIDR implant is removed on Day 7. Estrus can be detected from Day 8to Day 11. Further examples of synchronization methods include thecombination of gonadotropins and prostaglandin. In the so-called “SelectSynch protocol” (See Lamb G C, et al, Effectiveness of Select Synch(Gonadotropin-Releasing Hormone and Prostaglandin F2α) for SynchronizingEstrus in Replacement Beef Heifers, The Professional Animal Scientist,20:27-33 (2004), incorporated by reference in its entirety), aninjection of gonadotropin is administered on Day 0 with an injection ofprostaglandin administered on Day 7. Estrus typically occurs 36 hourbefore and up to 6 days after the prostaglandin injection. The “SelectSynch protocol” can be further modified to include a furthergonadotropin injection 2 days after the prostaglandin injection.

Accordingly, one embodiment of the present methodology entailssynchronizing a number of cows, detecting estrus in the synchronized cowpopulation, and then detecting the presence of a corpus luteum in cowsthat are estrus-negative.

By increasing the numbers of cows identified as receptive to embryotransfer and subsequent pregnancy, the present invention also increasesthe number of lactating cows that correspondingly increases the quantityof milk that can be produced per unit time. Dairy cattle such asHolstein have been repeatedly and selectively bred for increased milkproduction, and such cows can be used in the context of the presentinventive technology. While milk production level varies from breed tobreed, one cow can typically produce around 15,000 to 25,000 lbs of milkper lactation period. Ten or more lactations are possible during a cow'slifetime. But most cows are culled after two lactations, mainly for fourreasons: (1) infertility; (2) persistent and potentially fatal mammarygland infection; (3) persistent foot infection or leg problems causinginfertility and loss of production; and (4) failure to produce economiclevels of milk to justify their feed costs. The present inventiontherefore addresses the number one reason for culling dairy cows andmaximizes the efficiency of the rate of pregnancy by identifyingadditional cows receptive to embryo transfer, and also maximizes theefficiency of milk production.

It was shown as disclosed elsewhere herein, that estrus was synchronizedusing a single intramuscular injection of Lutalyse injectableprostaglandin into cows, and estrus detected by observing tail chalkonce daily. Fresh in vitro produced (IVP) embryos at Day 6.5 and 7.5,were then transferred to cows observed to be in estrus 11 days afterLutalyse injection on cow estrous cycle Days 6-10. Those cows that didnot show signs of estrus were then subsequently palpated for a corpusluteum (CL). If a CL was detected an IVP embryo was transferred.Pregnancy was detected by palpation at approximately 40 days.Significantly, the cows which did not show estrus but did have a corpusluteum, had a conception rate of 38.7% (FIG. 1 and Table 1).

Similarly, in the same study disclosed herein, thirty-one additionalcows were identified through CL palpation for embryo transfer whichincreased the total number of embryos transferred by 26%. This studyshows that these cows, which would have remained undetected, werecapable of becoming pregnant with an IVP embryo.

Furthermore, embryos have the unique ability to speed up or delaydevelopment in order to fit the stage of the uterine environment.Therefore the embryo and the uterus do not need to be in exact synchronyfor a pregnancy to occur. This cannot be said for artificialinsemination (AI). When AI occurs, it must be done within a very shorttime period (12 hour window). There are some protocols for timing AI soheat detection is unnecessary. However, these protocols require givingnumerous injections and can take several weeks from beginning of theprotocol to AI. If the service is unsuccessful repeating the protocolagain takes many additional weeks.

Because the corpus luteum structure does not exist on the ovary untilday 4-5 of the estrous cycle, 4-5 days after the necessary time forartificial insemination, the process of palpating a corpus luteumstructure after synchronization is not applicable for artificialinsemination programs. However, corpus luteum palpation is effective inembryo transfer, as the embryo is transferred on days 6-10 of theestrous cycle. The results from this study, for instance, as explainedin detail in the Examples, showed that the conception rates of cowswhich did not show signs of estrus but had a corpus luteum, and in whichan embryo was transferred, were not different than cows that wereobserved in estrus and known to be synchronized.

Thus, this new protocol will save dairy producers money insynchronization costs, as well as increasing the efficiency of obtainingpregnant animals, such as cows, that had not shown signs of estrus.Although these animals were capable of becoming pregnant they would nothave been identified using current, existing protocols.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein, and the laboratory procedures described herein, are thosewell known and commonly employed in the art. Standard techniques areused for mammal breeding and maintenance, analytical chemistry,molecular biology, enzymatic assays and pharmaceutical formulation anddelivery. Generally, enzymatic reactions and purification and/orisolation steps are performed according to the manufacturers'specifications.

The following examples serve to illustrate various embodiments of thepresent invention and should not be construed, in any way, to limit thescope of the invention.

All references cited herein, including patents, patent application andpublications, are hereby incorporated by reference in their entireties,where previously specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations and conditions, withoutundue experimentation. This application is intended to cover anyvariations, uses, or adaptations of the invention, following in generalthe principles of the invention, that include such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

EXAMPLES Example 1

Between July 2010 and January 2011 one hundred forty-eight Holstein,Holstein crossbred and Jersey cows were identified as repeat breeders bybeing over 120 days in milk or having had over 4 unsuccessful artificialinsemination services. Estrus was synchronized using a singleintramuscular injection of Lutalyse injectable prostaglandin (25 mg,Pfizer, New York, N.Y.). Estrus was detected by observing tail chalkonce daily. Fresh in vitro produced (IVP) embryos, Day 6.5 and 7.5, weretransferred to cows observed in estrus 11 days after Lutalyse injectionon cow estrous cycle Days 6-10. Those cows that did not show signs ofestrus were palpated for a corpus luteum (CL). If a CL was detected anIVP embryo was transferred. Pregnancy was detected by palpation atapproximately 40 days.

Synchrony of cows and embryos were calculated as cow day minus embryoday and ranged from −0.5 to 2.5 Days. The highest proportions of animalswere 0.5 and 1.5 Days. (n=85). Thirty-one animals that received embryosbut did not show signs of estrus had an unknown synchrony. The overallDay 40 conception rate was 31.3% for animals in this study. Those cowswith unknown synchrony, which did not show estrus but did have a CL, hada conception rate of 38.7% (FIG. 1 and Table 1).

In this study, dairy cattle were synchronized with one shot of Lutalyse.Lutalyse (prostaglandin) is able to cause regression of the corpusluteum (CL) on days 5 through 17 of the estrous cycle when a fullyfunctional CL is present on the ovary. Luteolysis is the regression ofthe CL which allows onset of estrus. Those animals past day 17 of thecycle would have natural CL regression if a pregnancy had not beenestablished. Only those animals between days 0 and 5 would not besynchronized. Therefore, on average the percentage of animals that wouldbe synchronized for estrus within the next 5 days would be 76.2%. Manyof these animals, however, would not show or may not be seen in heat. Inthis study tail chalk was used to detect estrus by once dailyobservation. The animals that were not detected were palpated for a CL.If a CL was present an embryo was transferred. Thirty-one additionalanimals were identified for embryo transfer which increased the totalnumber of embryos transferred by 26%. This study shows that thoseundetected animals were capable of becoming pregnant with an IVP embryo.

Embryos have the unique ability to speed up or delay development inorder to fit the stage of the uterine environment. Therefore the embryoand the uterus do not need to be in exact synchrony for a pregnancy tooccur. This cannot be said for artificial insemination (AI). When AIoccurs it must be done within a very short time period (12 hour window).There are some protocols for timing AI so heat detection is unnecessary.However, these protocols require giving numerous injections and can takeseveral weeks from beginning of the protocol to AI. If the service isunsuccessful repeating the protocol again takes many weeks.

Because the CL structure does not exist on the ovary until day 4-5 ofthe estrous cycle, 4-5 days after the necessary time for artificialinsemination, the process of palpating a CL structure on the ovary postsynchronization is not applicable to AI programs. However, CL palpationis effective in embryo transfer, as the embryo is transferred on days6-10 of the estrous cycle. Results from this study showed that theconception rates of animals which did not show signs of estrus but had aCL, and in which an embryo was transferred, were not different thananimals that were observed in estrus and known to be synchronized. Thisnew protocol saves dairy producers money in synchronization costs, aswell as impregnating animals that had not shown signs of estrus.Although these animals were capable of becoming pregnant they would nothave been identified using other protocols. In addition, this newprotocol also provides a superior method for impregnating animals overthe standard timed artificial insemination method, because it permitsfewer shots and fewer drugs to be administered to animals over time.This protocol provides an alternative and preferred method to increasepregnancies and therefore lifetime milk production.

Synchrony of cows and embryos were calculated as cow day minus embryoday and ranged from −0.5 to 2.5 Days. The highest proportions of animalswere 0.5 and 1.5 Days. (n=85). Thirty-one animals that received embryosbut did not show estrus had an unknown synchrony. The overall Day 40conception rate was 31.3%. Those cows with unknown synchrony, which didnot show estrus but did have a CL, had a conception rate of 38.7%(FIG. 1) and Table 1 (no. of animals in each group and the % of thosethat conceived, P>0.1).

TABLE 1 Conception Synchrony Pregnant Open Total Rate −0.5 4 8 12 33.3%0.5 9 27 36 25.0% 1.5 15 33 48 31.3% 2.5 1 3 4 25.0% Unknown 12 19 3138.7% Total 41 90 131 31.3%

Example 2

FIG. 3 and Table 2 show the results from 490 IVF embryo transfers toolder beef recipients from January 2011 to present. Animals were givenone shot of Lutalyse and observed for signs of estrus. Those in standingheat showed behavioral estrus by standing to be jumped by anotheranimal. Another method used to identify animals in estrus is by paintingthe tail head of all cows synchronized. If the paint is rubbed off theanimal is recorded as having showed sign of estrus. By palpating for thepresence or absence of a corpus luteum (CL) we were also able to inferthat estrus had occurred when the CL is present. In vitro produced (IVP)embryos, Days 6.5, 7, 7.5 and 8, were transferred to bothestrus-positive cows (cows observed in heat or showing outward signs ofheat), and estrus-negative but CL-positive cows (cows not observed inheat or does not show outward signs of heat, but has CL present) 10 to12 days after Lutalyse injection on cow estrous cycle Days 6-10. Due toour synchronization efforts, the estrus is assumed to have occurred inthe time frame necessary for embryo transfer. Using CL palpation we wereable to identify 129 additional animals for embryo transfer. Of those,34 were confirmed pregnant at 30 days of gestation (26.4%). However,none of these animals would have had the opportunity to become pregnant.The drop in pregnancy rate is expected as the synchrony of the animalsuterus and the embryo are unknown, but the fact that pregnancies dooccur is significant.

TABLE 2 Estrus PREG OPEN Detection PREG OPEN TOTAL RATE RATE StandingHeat 106 107 213 49.8% 50.2% Tail Chalk 73 75 148 49.3% 50.7% CLPalpation 34 95 129 26.4% 73.6% TOTAL 213 277 490 43.5% 56.5%

Example 3

FIG. 4 and Table 3 shows the results from 138 IVF embryo transferred ata working dairy (Dairy A) to repeat breeder animals from July toDecember 2010. Animals were given one shot of Lutalyse and observed forsigns of estrus by looking for the absence of tail chalk. Twenty-oneadditional animals were identified for embryo transfer by using CLpalpation. Of those, nine were confirmed pregnant at 45 days ofgestation (42.9%). The 21 animals in the CL palpation group had notshown signs of estrus as the tail chalk had not been rubbed off the tailhead. These animals would not have had the opportunity to becomepregnant without the use of CL palpation and embryo transfer. Fresh invitro produced (IVP) embryos, Day 6.5 and 7.5, were transferred to bothestrus-positive cows (cows observed in heat or showing outward signs ofheat), and estrus-negative but CL-positive cows (cows not observed inheat or does not show outward signs of heat, but has CL present) 11 daysafter Lutalyse injection on cow estrous cycle Days 6-10

TABLE 3 Estrus PREG OPEN Detection PREG OPEN TOTAL RATE RATE Tail Chalk32 85 117 27.4% 72.6% CL Palpation 9 12 21 42.9% 57.1% TOTAL 41 97 13829.7% 70.3%

Example 4

FIG. 5 shows results of 861 IVF embryo transfers to beef and dairycattle from January 2010 to present. Using CL palpation we were able toidentify a significant number of estrus-negative cows for embryotransfer. Of those, 28.7% were confirmed pregnant at 30-45 days ofgestation. None of these animals would have had the opportunity tobecome pregnant if the embryos are only transferred to cows that wereidentified as “in heat”, or estrus-positive.

What is claimed is:
 1. A kit to increase milk production comprising: (A)at least one corpus luteum detecting reagent or device; (B) at least onecow embryo to be transferred to at least one cow; and (C) instructionsfor kit use, wherein instructions comprise: i) identifying one or moreestrus-negative cows, wherein each cow is estrus negative if that cow isnot in heat or does not show outward signs of heat, and wherein said cowhas not been subjected to a timed embryo transfer procedure; ii)maintaining said one or more estrus-negative cows and testing for thepresence of a corpus luteum using the at least one corpus luteumdetecting reagent or device; iii) transferring the at least one cowembryo to at least one of the estrus-negative cows that has beenidentified by the testing to have a corpus luteum; and wherein use ofthe kit increases the number of pregnant cows, thereby increasing milkproduction compared to a cow population wherein the kit is not used. 2.A kit to increase milk production, comprising: (A) at least one corpusluteum detecting reagent or device; (B) at least one frozen cow embryoto be transferred to at least one cow; (C) at least one warming orthawing medium; (D) at least one transfer medium; and (E) instructionsfor kit use, wherein instructions comprise: i) identifying one or moreestrus-negative cows, wherein each cow is estrus negative if that cow isnot in heat or does not show outward signs of heat, and wherein said cowhas not been subjected to a timed embryo transfer procedure; ii)maintaining said one or more estrus-negative cows and testing for thepresence of a corpus luteum using the at least one corpus luteumdetecting reagent or device; iii) transferring via the at least onewarming or thawing medium and the at least one transfer medium, the atleast one frozen cow embryo to at least one of the estrus-negative cowsthat has been identified to have a corpus luteum using the at least onecorpus luteum detecting reagent or device; and wherein use of the kitincreases the number of pregnant cows, thereby increasing milkproduction compared to a cow population wherein the kit is not used. 3.A kit to increase milk production comprising: (A) at least one corpusluteum detecting reagent or device; (B) at least one cow embryo to betransferred to at least one cow; and (C) instructions for kit use,wherein instructions comprise: i) identifying one or moreestrus-negative cows, wherein each cow is estrus negative if that cow isnot in heat or does not show outward signs of heat, and wherein said cowhas not been subjected to a timed embryo transfer procedure; ii)maintaining said one or more estrus-negative cows and testing for thepresence of a corpus luteum using the at least one corpus luteumdetecting reagent or device; iii) transferring the at least one cowembryo to at least one of the estrus-negative cows that has beenidentified to have a corpus luteum using the at least one corpus luteumdetecting reagent or device; and wherein use of the kit increases thenumber of pregnant cows, thereby increasing milk production compared toa cow population wherein the kit is not used.